We have been investigating the relationship between the molecular pathways that underlie embryogenesis and those that are deregulated in carcinogenesis using the mammalian prostate as a model system. Our studies of the Nkx3.1 homeobox have elucidated its functions for development and carcinogenesis of the prostate. We have found that Nkx3.1 mutant mice display defects in prostatic epithelial differentiation and are predisposed to prostate cancer initiation. Moreover, loss-of-function of Nkx3.1 collaborates with that of the Pten and p27 Kip_ tumor suppressor genes in prostate carcinoma in compound mutant mice (i.e., Nkx3.1+/-; Pten+/-; p27+/-). Finally, epigenetic loss of Nkx3.1 protein (but not its mRNA) is a hallmark of prostate carcinogenesis, and loss of its expression is accompanied by upregulation of androgen receptor. Thus, we have hypothesized that Nkx3.1 represents a key regulator of prostatic epithelial differentiation as well as a modulator of androgen signaling. We further hypothesize that cancer susceptibility due to Nkx3.1 loss-of-function is a consequence of defects in prostatic epithelial differentiation and androgen signaling. We will now investigate: (Aim 1) The functions of Nkx3.1 in specification of prostatic epithelial differentiation using a tissue recombination approach, which provides a model system for studying epithelial and mesenchymal tissue requirements for prostatic growth and carcinogenesis. (Aim 2) The relationship of Nkx3.1 and androgen receptor for prostate differentiation and carcinogenesis by investigating: (i) the coordinate expression of Nkx3.1 and androgen receptor in the prostate; (ii) the role of the androgen receptor as a downstream mediator of Nkx3.1 function in prostate differentiation and carcinogenesis; and (iii) the consequences of altered androgen signaling for prostate carcinogenesis in mutant mouse models. (Aim 3): The mechanisms underlying the cooperativity and tissue-specificity of loss-of- function of Nkx3.1, Pten, and p27 kip1 prostate carcinogenesis, by studying the contributions of candidate effectors as well as pursuing exploratory approaches to identify novel mediators of Nkx3. I, Pten, and p27 kip1 cooperativity. (Aim 4) The regulation of Nkx3.1 protein expression in prostate development and cancer, by defining the regulatory sequences controlling Nkx3.1 protein expression, with the ultimate goal of developing a mouse model that lacks this mode of regulating Nkx3.1 protein expression. These mice will provide a valuable model for exploring the reversibility of prostate carcinogenesis in vivo.